Protein Biomarkers for Invasive Aspergillosis Diagnostics

描述（申请人提供）：随着易感人群的增加，烟曲霉引起的疾病负担大幅增加。此外，由于诊断困难，实际受影响人数可能被严重低估。侵袭性曲霉病患者预后的改善与早期诊断直接相关，这强调了更准确的诊断测试的必要性。诊断分析面临的挑战包括：该生物体无处不在，因此需要有能力区​​分污染、感染和疾病；二.易感宿主多种多样，疾病的发病机制可能有所不同；三.疾病综合症的范围，从过敏介导的过程到曲霉瘤再到侵袭性疾病。目前，诊断开发的大部分工作集中在碳水化合物抗原的血清检测、基于 PCR 的真菌负荷指标或用于检测抗真菌抗体反应的免疫分析上。真菌蛋白质组分析通常仅限于体外生长条件。该申请提议 Marta Feldmesser 博士（阿尔伯特·爱因斯坦医学院）与 Drs. 之间的合作。 John Galgiani 和 Vicki Wysocki（亚利桑那大学）在中性粒细胞减少和皮质类固醇治疗的小鼠动物模型中鉴定出与侵袭性疾病相关的特定蛋白质生物标志物，但在过敏性支气管肺曲霉病模型中未发现这些标志物。将探测真菌蛋白质组中的一系列标记物，从而使用质谱方法进行生物标记物鉴定，从而获得更灵敏和更特异的疾病标记物。然后重组蛋白将用于创建可用于基于质谱的测定开发的单克隆抗体。提出了六个具体目标： 1. 鉴定在侵袭性肺病期间而不是在真菌性哮喘期间特异存在的烟曲霉蛋白； 2. 表达并纯化侵袭性曲霉菌病的三个候选生物标志物的重组蛋白； 3. 制备目标 1 中鉴定的真菌蛋白的单克隆抗体，用于诊断测定； 4. 使用质谱法鉴定用于免疫测定的单克隆抗体； 5. 开发原型 ELISA，以检测实验性侵袭性和过敏性曲霉病小鼠模型以及来自其他 NIH 合同资源的临床标本中的候选生物标志物； 6. 开始将重组蛋白和 MAb 试剂迁移到多重和即时护理平台以进行临床应用。前两个目标将在奖励的 R21 部分期间完成，而其余四个目标将在 R33 期间完成。这些研究的完成将提供新颖的诊断方法，具有灵活应对特定临床环境的潜力。

DESCRIPTION (provided by applicant): With the rise in susceptible individuals, the burden of disease due to Aspergillus fumigatus has increased substantially. In addition, because of the difficulties in diagnosis, the actual number of affected people likely has been severely underestimated. Improved outcome in patients with invasive aspergillosis has been directly linked to earlier diagnosis, underscoring the need for more accurate diagnostic tests. Among the challenges posed for diagnostic assays include: i. the ubiquity of the organism, necessitating the ability to distinguish between contamination, infection, and disease; ii. the variety of susceptible hosts, in whom the pathogenesis of disease may vary; iii. the spectrum of disease syndromes, which range from allergy-mediated processes to aspergilloma to invasive disease. At present, the majority of effort in diagnostic development has focused on serum detection of carbohydrate antigens, PCR-based indicators of fungal burden or immunoassays for detection of anti-fungal antibody responses. Fungal proteomic analyses generally have been limited to in vitro growth conditions. This application proposes a collaboration between Dr. Marta Feldmesser (Albert Einstein College of Medicine) with Drs. John Galgiani and Vicki Wysocki (University of Arizona) to identify specific protein biomarkers associated with invasive disease in animal models of neutropenic and corticosteroid-treated mice, but that are not found in a model of allergic bronchopulmonary aspergillosis. The fungal proteome will be probed for the range of markers that will allow for more sensitive and specific markers of disease using mass spectrometry approaches to biomarker identification. Recombinant proteins then will be used to create monoclonal antibodies that can be used for mass spectrometry-based assay development. Six specific aims are proposed: 1. To identify A. fumigatus proteins present specifically during invasive pulmonary disease and not during fungal asthma; 2. to express and purify recombinant proteins of three candidate biomarkers of invasive aspergillosis; 3. to make monoclonal antibodies to fungal proteins identified in Aim 1 for use in diagnostic assays; 4. to use mass spectrometry to identify monoclonal antibodies for use in immunoassays; 5. to develop a prototype ELISA to detect candidate biomarkers in experimental invasive and allergic aspergillosis murine models and in clinical specimens from other NIH contract resources; and 6. to begin to migrate the recombinant protein and MAb reagents to multiplex and point-of-care platforms for clinical applications. The first two Aims would be accomplished during the R21 portion of the award, while the remaining four would be done during the R33 period. The accomplishment of these studies would provide novel diagnostic methods with potential for flexibility in response to particular clinical settings.